The present invention relates generally to the field of electrophysiology, wherein electrical measurements are made on biological cells and cell membranes to understand interactions between specific membrane components such as ion channels and transporters. Such measurements may be performed on living cells, membranes, or vesicles, as well as artificial membranes. More particularly, the invention resides in methods and apparatus enabling electrophysiological measurements to be made in parallel, without direct human intervention, thereby enhancing the cost effectiveness, throughput and general applicability of the technique to pharmaceutical drug screening and other procedures.
The electrical behavior of cells and cell membranes is of profound importance in basic research as well as in modern drug development. A specific area of interest in this field is in the study of ion channels and transporters [1]. Ion channels are protein-based pores found in the cell membrane that are responsible for maintaining the electro-chemical gradients between the extra cellular environment and the cell cytoplasm. Quite often these membrane channels are selectively permeable to a particular type of ion, e.g. potassium or sodium. The channel is generally comprised of two parts; the pore itself, and a switch mechanism that regulates the conductance of the pore. Examples of regulation mechanisms include changes in transmembrane voltage or the activation or deactivation of a membrane receptor via a chemical ligand. Ion channels are passive elements in that once opened, ions flow in the direction of existing chemical gradients. Ion transporters are similar in that they are involved in the transport of ions across the cell membrane, however they differ from ion channels in that energy is required for their function and they tend to actively pump against established electrochemical gradients.
Ion channels are prevalent in the body and are necessary for many physiological functions including the beating of the heart, voluntary muscle contraction and neuronal signaling. They are also found in the linings of blood vessels allowing for physiological regulation of blood pressure and in the pancreas for control of insulin release. As such, the study of such channels is a very diverse and prolific area encompassing basic academic research as well as biotech and pharmaceutical research. Experiments on ion channels are typically performed on cell lines with endogenously express the ion channel of interest (xe2x80x9cnative channelsxe2x80x9d) as well as on recombinant expression systems such as the Xenopus Oocyte or mammalian cell lines (e.g. CHO, HEK etc.) where the channels are inserted by well-known transfection techniques [2] [3]. Electrophysiology is also performed on isolated cell membranes or vesicles as well as in synthetic membranes where solubilized channels are reconstituted into a manufactured membrane [4].
To date, the most useful and widely utilized tool for the study of ion channels and transporters is via a technique called patch clamping. This technique was first introduced almost 25 years ago [5] [6] [7], and consists of using a small glass capillary to function as an electrode in measuring currents and voltages from individual cells. FIG. 1 depicts a typical patch clamp measurement geometry. A glass capillary 2 is first heated and pulled to a fine tip. The capillary is then filled with a saline buffer solution 4 and fitted with a Ag/AgCl electrode 6. The function of the Ag/AgCl electrode is to provide an electrical connection to a wire via the reversible exchange of chloride ions in the pipette solution.
Through the use of a microscope and micromanipulating arm (not shown), the user finds a biological cell or cell membrane 8 containing ion channels 10 of interest and gently touches the cell membrane with the pipette. The measurement circuit is complete via the external ionic solution 12 and a second Ag/AgCl bath electrode 14. A high-impedance operational amplifier 16 senses the current flowing in the circuit which is subsequently recorded and analyzed with a data recording system 18. The key to the function of the technique is the ability to form a high electrical resistance (xcx9c1Gxcexa9) seal between the glass pipette and the cell membrane 20, so that the current recorded by the amplifier is dominated by ions 22 flowing through the cell membrane and not ions flowing around the glass pipette directly into the bath solution.
Once a high-resistance seal is achieved between the pipette and the cell membrane, there are many measurement configurations that the system can take, the scope of which is beyond this disclosure. One of the more common is the whole cell voltage clamp. In this configuration it is necessary to permeabilize the portion of membrane at the end of the pipette 24 so as to effectively place the pipette electrode inside the cell. This in turn allows for an external voltage command 26 to be placed between the intracellular pipette electrode and the extracellular bath electrode, thereby providing control of the cell""s transmembrane voltage potential. The term xe2x80x9cwhole cellxe2x80x9d is derived from the fact that with this configuration, the instrument measures the majority of the currents in the entire cell membrane.
The electrical permeabilization of the membrane at the end of the pipette can be induced in many ways but is often achieved by voltage pulses of sufficient strength and duration such that the membrane inside the pipette physically breaks down. This is commonly referred to as xe2x80x9czappingxe2x80x9d [8] and is a well-known technique in the field. Another technique utilized to electrically permeabilize the membrane is through the use of certain antiobiotics such as Nystatin and Amphotericin B [9]. These chemicals work by forming chemical pores in the cell membrane that are permeable to monovalent ions such as chloride. Since chloride is the current carrying ion for the commonly used Ag/AgCl electrode, these antiobiotics can produce a low resistance electrical access to the interior of the cell. The advantage of the chemical technique is that the membrane patch remains intact such that larger intracellular molecules remain inside the cell and are not flushed out by the pipette solution as with the zapping technique. The use of chemicals to electrically permeabilize the membrane is also a commonly used technique in the field and is referred to as a xe2x80x9cperforated patchxe2x80x9d [8] [9][10].
The formation of the high-resistance electrical seal enables the measurement system to detect very small physiological membrane currents, (e.g. 10xe2x88x9212 amp). In addition, by perforating a portion of the cell membrane either electrically or chemically, it possible to control the voltage (voltage clamp) or current (current clamp) across the remaining intact portion of the cell membrane. This greatly enhances the utility of the technique for making physiological measurements of ion channel/transporter activity since quite often this activity is transmembrane voltage dependent. By being able to control the trans-membrane voltage (or current), it is possible to stimulate or deactivate ion channels or transporters with great precision and as such greatly enhance the ability to study complex drug interactions.
The development of the patch clamp technique revolutionized the field allowing for the direct electrical measurement of ion channel/transporter events in living cells, cell membranes and artificial membranes. However, existing patch clamp techniques require the use of a skilled operator using a microscope and micromanipulating arm to record data from a single cell or membrane preparation using a small glass capillary. Typically, a recording session may take tens of minutes to complete and requires a high level of dexterity by the operator. In addition, especially in the case of drug screening, it is generally preferable to obtain a new cell sample for each different chemical entity to be tested. As such, the technique is not relevant to looking at thousands of different conditions (e.g. chemical stimuli) per day, a common need in the biotech or pharmaceutical industry.
U.S. Pat. No. 6,063,260 to Olesen describes a system intended to improve the throughput and decrease the fluid volume required of standard patch clamp technology. The improvement relies on using a standard HPLC autosampler apparatus integrated into a standard patch clamp arrangement in order to more easily inject multiple fluids samples into the measurement system. The invention claims to increase throughput by making multiple sequential fluid additions to the same biological membrane faster and easier. However, the Oleden invention is deficient in several respects. First, it does not allow for a plurality of different biological samples to be measured simultaneously. In addition, it does not eliminate the labor intensive aspects of micromanipulation involved in standard patch clamp electrophysiology, nor does it address cases in biological drug screening where multiple chemical reagent additions to the same biological sample are to be avoided (as in the case of high-throughput drug screening).
Published PCT Application WO 99/66329 discusses the use of a perforated screen to conduct tests on biological materials, but is clear from the disclosure that the proposed system presents significant severe limitations in terms of a practical implementation. First, all embodiments discussed in the WO 99/66329 application utilize multiple apertures per fluid well, placing reliance on the growth of confluent cell matrices to effectuate sealing of the multiple perforations formed in relatively thick material. In addition, although the published application makes reference to automation, no workable, fully integrated systems are disclosed which are capable of high throughput and reliability.
In broad and general terms, the invention described enables electrophysiological measurements to be made more quickly than with standard patch clamp techniques. The invention uses a thin, preferably layered substrate having a properly sized hole, on the order of a few microns in diameter, allowing a cell or biological membrane to be maneuvered by fluid flow to the hole independent of direct human intervention, thereby eliminating the use of a microscope and micromanipulating arm. This approach not only makes the measurement easier, it also provides a platform by which a plurality of measurements can be made simultaneously, greatly improving measurement throughput.
Through proper selection and processing of the substrate material, hole geometry, and attention to the way in which the biological membrane interacts with the substrate, a high-resistance electrical seal on the order of several hundred Mxcexa9 to 1 Gxcexa9 is achieved. Preferred substrates include thin plastic films in which small holes have been photomachined using a laser. These substrates were then vacuum deposited with thin layers of glass in order to aid in the formation of the high-resistance seal. Silicon substrates are also described, wherein standard photolithographic/wet etching techniques are to make the holes. In both cases, individual cells are then positioned onto isolated holes using differential pressure.
The invention further contemplates a substrate geometry which is directly applicable to the development of a high-throughput instrument whereby thousands of single cell electrophysiological recordings could be acquired in a single day. In addition, the invention encompasses an integrated electrophysiogical measurement system which includes a computer controlled data collection system, an integrated electronics head for making parallel electrical measurements, and an integrated fluidics head used in part to transfer test compounds into the measurement process.